Enveloped animal viruses attach to and enter their host cells via the interaction of certain viral proteins located in the virion membrane (envelope proteins) and host cell surface proteins (receptors and coreceptors for the virus). Receptor recognition and binding are mediated by the virus's envelope protein. Human Immunodeficiency Virus type 1 (HIV-1) gains entry into the human host cell by using CD4 and either CXCR4 (X4) or CCR5 (R5) coreceptors (Deng et al., 1996, Nature 381(6584):661-66). Virus entry is an attractive target for anti-viral treatment, and numerous drugs designed to block virus attachment or membrane fusion have been or are currently being evaluated in preclinical or clinical studies for HIV treatment (See, e.g., Richman, 1998, Nature Med., 4:1232-1233; PhRMA, 1999, “New Medicines in Development for AIDS,” Pharmaceutical Research and Manufacturers of America; Stephenson, 1999, JAMA, 282:1994). Some examples of entry inhibitors that have been or are being investigated include attachment inhibitor SCH-D (vivriviroc, which blocks the interaction between viral membrane proteins of HIV-1 and the cellular coreceptor CCR5, Schering-Plough), UK-427857 (maraviroc; Pfizer), TNX-355 (Tanox Inc.), AMD-070 (AnorMED), Pro 140 (Progenics), FP-21399 (EMD Lexigen), BMS-488043 (Bristol-Myers Squibb), and T-20 (enfuvirtide; Roche/Trimeris).
The effectiveness of the currently available drugs for treatment of HIV, however, varies from subject to subject depending, at least in part, on genetically-controlled susceptibility to each drug. Over 200 mutations have been identified as being associated with reduced susceptibility to one or more of the approved drugs (Clercq, 2009, Int. J. Antimicrob. Agents 33:307-20; Shafer and Schapiro, 2008, AIDS Rev. 10:67-84; Clavel and Hance, 2004, N. Engl. J. Med. 350:1023-35; Johnson, 2008, Topics in HIV Med. 16:138-45; Bennett, 2009, PLoS ONE 4:e4724). Due to the complexity of the treatment options available and the many resistance (reduced susceptibility) associated mutations, it is increasingly difficult to develop a comprehensive understanding of HIV drug resistance. Resistance mutations differ in their potency to resist drug pressure, in their degree of cross-resistance to different drugs or drug classes, and in the fitness costs induced in the absence of treatment. Moreover, their effects depend to varying degree on the context of accompanying mutations (Rhee et al., 2004, Antimicrob. Agents and Chemo. 48:31226; Bonhoeffer et al., 2004, Science 306:154750).
Drug resistance testing for individuals infected with HIV-1 is a key component of the management of antiretroviral therapy in North America and Europe. And in particular, accurate coreceptor tropism (CRT) determination is critical when making treatment decisions in HIV management. Assays for assessment of drug susceptibility are based on the sequencing of a patient's virus (genotyping), on virus replication inhibition in vitro (phenotyping), or both.
The HIV-1 envelope glycoprotein gp120 contains five highly variable regions or loops, designated V1 through V5, that are separated by four relatively “constant” regions (C1-C4). The first four variable regions form loops through intramolecular disulfide bonds. These variable regions are thought to cover a significant portion of the exposed surface on the trimeric gp120 complex. Gp120 has significant sequence variation, which may arise through recombination and point mutation, as well as by insertion and deletion of one or more nucleotides. The V1/V2 region and the V3 loop of the envelope protein are targets for neutralizing antibodies, and the V3 loop largely determines whether a virus uses R5, X4, or either coreceptor to infect its host cells. Given the use of entry inhibitors as a treatment option, it is critical to have diagnostic assays available that quickly and accurately determine the dominant coreceptor tropism in a clinical setting.
The variability of the amino acid sequence of the third hypervariable (V3) loop is shown in FIG. 1. Genotype based in silico prediction of virus tropism utilizing the sequence of the V3 loop of the envelope protein offers a rapid test for coreceptor usage. To date, many bioinformatics methods for tropism prediction have been developed and tested. These bioinformatics predictors include support vector machines (SVM) (Pillai et al., 2003, AIDS Res. Hum. Retrovir. 19(2):145-49; Sing et al., 2007, Antirviral Therapy 12(7):1097-1106), neural networks (NN) (Resch et al., 2001, Virology 288(1):51-62), decision trees (Masso and Vaisman, BMC Bioinformatics 11:494), position specific scoring matrices (PSSM) (Jenesen et al., 2003, J. Virol. 77(24):13376-88), multiple linear regression (Briggs et al., 2000, AIDS 14(18):2937-39), and the 11/25 rule (De Jong et al., 1992, J. Virol. 66(11):6777-80). However, many of these methods were trained on clonal sequences, and may not be adequate for tropism testing of clinical isolates that are often heterogeneous and have high level of sequence ambiguity (Low et al., 2007, AIDS 21(14):F17-24). Moreover, these methods generally are developed by fitting a model into the respective training set, and often do not perform as well with independent or unseen datasets (Jensen et al., 2003, AIDS Rev. 5(2):104-12).
What is needed, therefore, are efficient and accurate diagnostic assays and methods that can be used to quickly and accurately determine the dominant coreceptor tropism for a particular virus or population of viruses in order to guide patient treatment. Systems and computer readable media for use in such assays and methods are also needed.